title: Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.
abstract: Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.
authors.affiliation: Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Maass
authors.forename: Susanne
authors.initials: S
authors.lastname: Sievers
authors.forename: Daniela
authors.initials: D
authors.lastname: Zühlke
authors.forename: Judith
authors.initials: J
authors.lastname: Kuzinski
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Sappa
authors.forename: Jan
authors.initials: J
authors.lastname: Muntel
authors.forename: Bernd
authors.initials: B
authors.lastname: Hessling
authors.forename: Jörg
authors.initials: J
authors.lastname: Bernhardt
authors.forename: Rabea
authors.initials: R
authors.lastname: Sietmann
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.forename: Michael
authors.initials: M
authors.lastname: Hecker
authors.forename: Dörte
authors.initials: D
authors.lastname: Becher
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000166:cytology / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013211:Staphylococcus aureus / Q000166:cytology / Q000378:metabolism; D018511:Systems Integration
chemical_list: D001426:Bacterial Proteins; D011506:Proteins